It is the central tenant of this project and program that the most appropriate method that can be used to study the function of CRU proteins is to first remove them and then replace them in the context of a muscle cell. This method also makes it possible to mutate these CRU proteins thus facilitating the examination of critical protein-protein interactions. Core B will perform the repetitive tasks that are necessary to support all four Projects. This core will provide an important and integrated facility to receive gene targeting constructs from Project 1 (or elsewhere) and to create new constructs themselves. They will transfect these constructs into ES cells and identify homologously targeted clones. Core B will also coordinate with the Brigham and Women's Hospital Transgenic Mouse Facility to use these targeted ES cells to produce "knock out(in)" mice which in turn will provide animals to be studied by all four Projects. Core B will take cDNA constructs from Projects 1 and 3 and package them into HSV1 virions to be used for transient expression and distribute them to all 4 projects. They will take selected constructs and package them into AAV/HSV hybrid virions and use these virions to transduce appropriate myoblast cell lines, identify cells that have integrated the construct into their genome and expand these clones to be used for all 4 projects. Another major function of Core B will be to maintain the transgenic and heterozygous "knock out" animal lines and take responsibility for possible interbreeding to produce animals with a "knock out" of more than one protein, or animals in which the "knock out" phenotype is "rescued" by having wild-type or a mutated protein "knocked in". The uniform and consistent supply of exactly the same study models to all four Projects by this Core will allow the truly integrated approach to the study of e-c coupling.